Pattern of organotin inhibition of methanogenic bacteria.

Seven organotin compounds and tin chloride were tested for their effects on the methanogenic bacteria Methanococcus thermolithotrophicus, Methanococcus deltae delta LH, and Methanosarcina barkeri 227. The methanogens were strongly inhibited by triethyltin, tripropyltin, and monophenyltin compounds, generally at concentrations below 0.05 mM. Less inhibition by tributyltin and diphenyltin was observed at levels below 0.1 mM, but complete inhibition was observed at a 1 mM concentration. Tin chloride inhibited all methanogens, with nearly complete inhibition at a 1 mM concentration. There was no inhibition by tetra-n-butyltin and triphenyltin compounds even at 2 mM, the highest concentration tested. The 50 and 100% inhibitory concentrations of all compounds were estimated; these values varied with both the compound tested and the bacterium tested. The 50% inhibitory concentration estimate generally decreased (i.e., giving a higher toxicity) as the total surface area of the alkyltin molecules decreased. These results differ considerably from those reported previously for aerobic microorganisms (G. Eng, E. J. Tierney, J. M. Bellama, and F. E. Brinckman, Appl. Organometallic Chem. 2:171-175, 1988), where a clear correlation between increasing total molecular surface area and increasing toxicity was documented with a variety of organisms. Using the same procedures as for the methanogens, we examined the effects of organotin compounds on Escherichia coli growing aerobically or anaerobically. The E. coli inhibition pattern clearly resembled that seen in the data of Eng et al., under both aerobic and anaerobic conditions.     

Specific inhibition of sulphate-reducing bacteria in methanogenic co-culture

Various inhibitors of Desulfovibrio desulfuricans were examined for their selectivity in co-cultures with Methanobacterium formicicum . The inhibitors were chlorohexidine diacetate, m -dinitrobenzene, 2,4-dinitrophenol, potassium nitrate and sodium molybdate. Only sodium molybdate was able to inhibit D. desulfuricans selectively in the methanogenic co-culture. Sodium molybdate at all the concentrations used inhibited D. desulfuricans without adversely affecting M. formicicum .     

Methanopterin and methanogenic bacteria

Methanogenic bacteria comprise a selected group of microorganisms that derive their energy for growth from the hydrogen-dependent reduction of CO2 to methane or the disproportionation of reduced one-carbon compounds and acetate to CO2 and methane. In the reduction and oxidation steps at the formyl, hydroxymethyl and methyl level the one-carbon unit remains bound to the reduced form of methanopterin, a pterin derivative typical of methanogenic bacteria. In addition, the reduced methanopterin, 5,6,7,8-tetrahydromethanopterin, is involved in a number of anabolic reactions. Methanopterin is structurally and functionally the counterpart of folic acid found in other organisms. In this review the occurrence and properties of methanopterin and its derivatives, as well as the biosynthesis and the role in the different catabolic and anabolic reactions are discussed against the background of folic acid biochemistry.     

The biology of methanogenic bacteria

[This corrects the article on p. 517 in vol. 41.].     

Ether-containing lipids of methanogenic bacteria

Acid-hydrolysis of the phospholipid fraction of $\text{Methanobacterium thermoautotrophicum}\text{,}\text{Methanobacterium formicicum}$ and $\text{Methanospirillum hungatii}$ demonstrated the presence of two neutral lipid products. Characterization of these lipids resulted in their identification as dialkyl glyceryl ether and diglycerol tetraethers. The ether-linked alkyl chains were identified as the 20- and 40-carbon branched chains for the diether and tetraether, respectively. $\text{M}\text{.}\text{thermoautotrophicum}$ and $\text{M}\text{.}\text{formicicum}$ were also characterized by the presence of acid-stable phospholipid components.     

7-methylpterin in methanogenic bacteria

Abstract A blue fluorescent compound was extracted and purified from cells of Methanobacterium thermoautotrophicum . The compound was identified as 7-methylpterin on the basis of its (physico-) chemical properties and by comparison with 7-methylpterin prepared by organic synthesis. The compound is present in all methanogenic bacteria studied so far and it provides methanogenic bacteria the characteristic blue fluorescence observed upon fluorescence microscopy.     

Ribose biosynthesis in methanogenic bacteria

NMR spectroscopy was used to determine the labeling patterns of the ribose moieties of ribonucleosides purified from Methanospirillum hungatei, Methanococcus voltae, Methanobrevibacter smithii, Methanosphaera stadtmanae, Methanosarcina barkeri and Methanobacterium bryantii labeled with ¹³C-precursors. In most methanogens tested ribose was labeled in a manner consistent with the operation of the oxidative branch of the pentose phosphate pathway. In contrast, transaldolase and transketolase reactions typical of a partial nonoxidative pentose phosphate pathway are hypothesized to explain the different labeling patterns and enrichments of carbon atoms observed in the ribose moiety of Methanococcus voltae. The source of erythrose 4-phosphate needed for the transaldolase reaction proposed in Methanococcus voltae, and for biosynthesis of aromatic amino acids in methanogenic bacteria in general, was assessed. Phenylalanine carbon atom C-7 was labeled by [1-¹³C]pyruvate in Methanospirillum hungatei, Methanococcus voltae, and Methanococcus jannaschii, the only methanogens which incorporated sufficient label from pyruvate for testing. Reductive carboxylation of a triose precursor (derived from pyruvate) to synthesize erythrose 4-phosphate is consistent with the labeling patterns observed in phenylalanine and ribose.Abbreviation TCA Tricarboxylic acid Issued as NRCC Publication No. 37382     

Plasmid DNA from methanogenic bacteria

In total, 73 strains of methanogen isolates from our laboratory and 6 from culture collections were examined for the presence of plasmid DNA. Five strains were found to contain detectable plasmids. Multiple plasmids were found in two isolates, while three strains contained only one plasmid each. A physical map of the plasmid pT3 was constructed by use of six different restriction endonucleases. All sites were aligned with a single BgII site, and the position of the restriction sites was determined by double or sequential digestion of the plasmid DNA.     

Distribution of cytochromes in methanogenic bacteria

Abstract Various methanogenic bacteria belonging to the orders Methanobacteriales, Methanococcales , and Methanomicrobiales were examined for the presence of cytochromes. Those methanogens which are capable of growing only on H ₂+ CO ₂ or formate were found to lack cytochromes. However, membrane-bound cytochromes were detected in species able to utilize methanol, methylamines or acetate.     

Quantification of corrinoids in methanogenic bacteria

Corrinoids in several diverse species of methanogens were quantified by a bioassay utilizingEscherichia coli 113–3, a corrinoid auxotroph. All five species examined contained >0.65 nmol corrinoid/mg dry cells when grown on H₂/CO₂ as carbon and energy source. The highest corrinoid levels (4.1 nmol/mg cells) were found inMethanosarcina barkeri grown on methanol. The amount of corrinoids found in this species was dependent on growth conditions, but, regardless of energy source, metabolized levels inMethanosarcina barkeri were higher than those found in theMethanobacterium species examined (M. arbophilicum, M. formicium, M. ruminantium, andM. thermoautotrophicum).     

Genome complexity of methanogenic bacteria.

The genome complexities of different methanogenic bacteria were investigated by using an optical method to study renaturation kinetics of single-stranded DNA. The observed genome sizes ranged from 1.0 X 10(9) to 1.8 X 10(9) daltons, which is a typical range for procaryotic cells. Melting profiles of the DNA of three methanogenic species from different families show fractions which have a higher A . T content than the average DNA of that species.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

Distribution of polyamines in methanogenic bacteria

Members of all four families of methanogenic bacteria were analyzed for polyamine concentrations. High-performance liquid chromatography analysis of dansylated cell extracts revealed typical polyamine patterns for each family. Members of Methanobacteriaceae (family I) were characterized by very low polyamine concentrations; members of Methanococcaceae (family II) were characterized by putrescine and high spermidine concentrations; members of Methanomicrobiaceae (family III) were characterized by the presence of putrescine, spermidine, and sym-homospermidine; and members of Methanosarcinaceae (family IV) contained only high concentrations of sym-homospermidine in addition to putrescine. The highest polyamine concentration was found in Methanosarcina barkeri Jülich, with 0.35% putrescine in the dry cell material. The polyamine distribution found coincides with the dendrogram based on comparative cataloguing of 16S rRNA and offers a new, rapid chemotaxonomic method for characterizing methanogenic bacteria. Variation of the growth substrates (H2-CO2, methanol, acetate, and trimethylamine) for M. barkeri resulted in quantitative but not qualitative differences in polyamine composition.     

One carbon metabolism in methanogenic bacteria

Two strains of Methanosarcina (M. Barkeri strain MS, isolated from sewage sludge, and strain UBS, isolated from lake sediments) were found to have similar cellular properties and to have DNA base compositions of 44 mol percent guanosine plus cytosine. Strain MS was selected for further studies of its one-carbon metabolism. M. barkeri grew autotrophically via H₂ oxidation/CO₂ reduction. The optimum temperature for growth and methanogenesis was 37°C. H₂ oxidation proceeded via an F₄₂₀-dependent NADP⁺-linked hydrogenase. A maximum specific activity of hydrogenase in cell-free extracts, using methyl viologen as electron acceptor, was 6.0 mol min · mg protein at 37°C and the optimum pH (9.0). M. barkeri also fermented methanol andmethylamine as sole energy sources for growth. Cell yields during growth on H₂/CO₂ and on methanol were 6.4 and 7.2 mg cell dry weight per mmol CH₄ formed, respectively. During mixotrophic growth on H₂/CO₂ plus methanol, most methane was derived from methanol rather than from CO₂. Similar activities of hydrogenase were observed in cell-free extracts from H₂/CO₂-grown and methanol-grown cells. Methanol oxidation apparently proceeded via carrierbound intermediates, as no methylotrophy-type of methanol dehydrogenase activity was observed in cell-free extracts. During growth on methanol/CO₂, up to 48% of the cell carbon was derived from methanol indicating that equivalent amounts of cell carbon were derived from CO₂ and from an organic intermediate more reduced than CO₂. Cell-free extracts lacked activity for key cell carbon synthesis enzymes of the Calvin cycle, serine path, or hexulose path.Abbreviations CAPS cycloaminopropane sulfonic acid - CH₃-SCoM methyl coenzyme M - DCPIP 2,6-dichlorophenolindophenol - DEAE diethylaminoethyl - dimethyl POPOP 1,4-bis-2-(4-mothyl-5-phenyloxazolyl)-benzene - DNA deoxyribonucleic acid - dpm dismtegrations per min - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - F₄₂₀ factor 420 - G+C guanosine plus cytosine - NAD⁺ nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - PBBW phosphate buffered basal Weimer - PMS phenazine methosulfate - PPO 2,5-diphenyloxazole - rRNA ribosomal ribonucleic acid - RuBP ribulose-1,5-bisphosphate - Tris tris-hydroxymethyl-aminomethane - max maximum specific growth rate     

Association of methanogenic bacteria with rumen ciliates

In 11 species of rumen ciliates belonging to nine genera of the family Ophryoscolecidae (order Entodiniomorphida) an ectosymbiosis with methanogenic bacteria was found. The bacteria could be identified as methanogens on the basis of the presence of specific fluorescent coenzymes (F350 and F420). This somatic interaction may reflect a metabolic interaction in which efficient interspecies hydrogen transfer benefits both partners.     

Antibody analysis of relationships among methanogenic bacteria

A bank of antisera to the majority of methanogenic bacteria is now available. Three antibody probes, R, S, and T, were derived from each antiserum in the bank and used for analysis of antigenic relatedness among methanogens by immunofluorescence. The T probe reacted only with the immunizing (or homologous) strain, the S probe gave strong cross-reactions with strains of the same species, and the R probe revealed some interspecies relationships. The results were confirmed and extended by enzyme immunoassays and standard serological methods involving serial dilution analysis, cross-adsorptions, and the use of reference strains. The immunological methods and standardized antibody probes are useful for rapid identification of methanogens and measurements of antigenic relationships which aid in the classification of these bacteria.